Method of making demineralized bone particles

ABSTRACT

Demineralized bone particles are obtained by demineralizing whole bone and thereafter subdividing the demineralized bone to provide the demineralized bone particles.

BACKGROUND OF THE INVENTION

This invention relates to a method of making demineralized boneparticles useful in, or as, implants having a variety of orthopedicapplications. More particularly, this invention relates to a method ofmaking particles from demineralized bone that results in a greater yieldof demineralized bone particles than that provided by prior art methodsof producing such particles.

The manufacture of demineralized bone particles and compositions,materials and devices containing demineralized bone particles and theiruse in the repair of bone defects and for other orthopedic applicationsare known.

The microstructure of cortical bone consists of bundles, or fibers, ofmineralized collagen that are oriented parallel to the long axis of theknown methods for making demineralized bone particles involvesubdividing sections of whole, i.e., mineralized, bone, e.g., by suchmechanical operations as shredding, milling, shaving, machining, etc.,to provide particles which are then demineralized, e.g., by treatmentwith acid. The resulting demineralized bone particles exhibitosteoinductive properties that make them useful as, or in, implantsintended for use in bone repair and other orthopedic applications. Onedrawback of known methods of making demineralized bone particles is thatonly a portion of the bone stock, e.g., 45-65% by weight, will yielddemineralized bone particles. In addition, because of the mechanicallimitations of the bone milling machinery, e.g., the need to grip thebone stock in the jaws of the machine, only donor bone of a fairlysubstantial size, e.g., intact cortical shafts, can be used as to thesource of the demineralized bone particles.

The limited amount of demineralized bone particles that is obtained bythe prior art methods is of concern due to the limited availability ofdonor bone. At this time, regulations do not permit the pooling of donorbone material. Since the quantity of demineralized bone particles thatcan be obtained is limited both by the availability of donor bone andthe size of the bone, there is a need for a method of makingdemineralized bone particles that is not subject to the constraintsimposed by these limiting factors.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method of makingdemineralized bone particles which makes optimum use of donor bone.

It is a further object of the invention to provide a method of makingdemineralized bone particles that results in a greater yield ofparticles for a given quantity of whole bone compared to that providedby prior art methods.

It is yet another object of the invention to provide demineralized boneparticles in the form of fibers or fibrous bundles of bone collagen byapplication of mechanical pressure to demineralized bone stock.

Further objects of the invention will be apparent to those skilled inthe art in view of the above objects and the foregoing specification.

In keeping with these and related objects of the invention, there isprovided a method of making demineralized bone particles which comprisesdemineralizing whole bone and thereafter subdividing the demineralizedbone into demineralized bone particles.

In general, the yield of demineralized bone particles obtained by themethod of this invention is significantly greater, e.g., from about 5 toabout 20 wt. % greater, than that obtained by first subdividing thewhole bone into mineralized bone particles and only thereafterdemineralizing the mineralized bone particles to provide demineralizedbone particles.

The term “particles” as utilized herein is intended to includerelatively small bone pieces such as fibers, bundles of looselyconnected fibers, threads, narrow strips, thin sheets, chips, shards,powders, etc., that possess regular, irregular or random geometries andwhich may, or may not be, completely separated from each other.

The expression “whole bone” as utilized herein refers to bone thatcontains its full naturally occurring mineral content and includesanatomically complete bones and sections thereof.

The term “demineralized” as used herein refers to bone containing lessthan about 95% of its original mineral context. The expression “fullydemineralized” as used herein refers to bone containing less than about5% of its original mineral context.

The terms “osteogenic” as used herein shall be understood to refer tothe ability of a material or substance to induce new bone formation viathe participation of living cells from within the substance and“osteogenesis” as the mechanism or result.

The terms “osteoinductive” as used herein shall be understood to referto the ability of a material or substance to recruit cells from the hostwhich have osteogenic potential and the ability to form ectopic bone and“osteoinduction” as the mechanism or result.

The terms “osteoconductive” as used herein shall be understood to referto the ability of a material or substance or material to providesurfaces that are receptive to the growth of new host bone and“osteoconduction” as the mechanism or result.

The terms “autogenic”, “allogenic” and “xenogenic” are used hereinrelative to the ultimate recipient of the bone tissue.

DETAILED DESCRIPTION OF THE INVENTION

The whole bone suitable for making the demineralized bone particles ofthis invention can be donor bone from any source. Thus, autogenic,allogenic or xenogenic bone can be used with autogenic and allogenicbone being preferred. An especially useful source of xenogenic tissuecan be porcine, equine, or bovine. The bone can be cortical, cancellousor corticocancellous. The preferred bone is cortical allogenic bone,e.g., femur, tibia, fibula, radius, ulna, etc.

The method of this invention is applicable to whole bone in a variety ofsizes. Therefore, the bone utilized as the starting, or stock, materialwill range in size from relatively small pieces of bone to bone of suchdimensions as to be recognizable as to its anatomical origin. Ingeneral, the pieces or sections of whole bone stock can range from about1 to about 400 mm, and preferably from about 5 to about 100 mm, inmedian length, from about 0.5 to about 20 mm, and preferably from about2 to about 10 mm, in median thickness and from about 1 to about 20 mm,and preferably from about 2 to about 10 mm, in median width.

After the bone is obtained from the donor, it is processed, e.g.,cleaned, disinfected, defatted, etc., using methods well known in theart. The entire bone can then be demineralized or, if desired, the bonecan just be sectioned before demineralization. The entire bone or one ormore of its sections is then subjected to demineralization in order toreduce the inorganic content to a low level, e.g., to contain less thanabout 10% by weight, preferably less than about 5% by weight and morepreferably less than about 1% by weight, residual calcium.

Demineralization of the bone can be accomplished in accordance withknown and conventional procedures. Demineralization procedures removethe inorganic mineral component of bone by employing acid solutions.Such procedures are well known in the art, see for example, Reddi etal., Proceeding of the National Academy of Sciences of the United Statesof America 69, pp. 1601-1605 (1972), incorporated herein by reference.The strength of the acid solution, the shape and size of the bone andthe duration of the demineralization procedure will determine the extentof demineralization. Generally speaking larger bone portions as comparedto small particles will require more lengthy and vigorousdemineralization. Guidance for specific parameters for thedemineralization of different size bone can be found in U.S. Pat. No.5,846,484, Harakas, Clinical Orthopaedics and Related Research, pp239-251(1983) and Lewandrowski et al., Journal of Biomedical MaterialsResearch, 31, pp. 365-372 (1996), each of which is incorporated byreference herein.

In a demineralization procedure useful in the practice of the inventionherein, the bone is subjected to a defatting/disinfecting step that isfollowed by an acid demineralization step. A usefuldefatting/disinfectant solution is an aqueous solution of ethanol, theethanol being a good solvent for lipids and the water being a goodhydrophilic carrier to enable the solution to penetrate more deeply intothe bone particles. The aqueous ethanol solution also disinfects thebone by killing vegetative microorganisms and viruses. Ordinarily atleast about 10 to about 40 weight percent by weight of water (i.e.,about 60 to about 90 weight percent of defatting agent such as alcohol)should be present in the defatting/disinfecting solution to produceoptimal lipid removal and disinfection within the shortest period oftime. A useful concentration range of the defatting solution is fromabout 60 to 85 weight percent alcohol or about 70 weight percentalcohol. An alternative or supplemental defatting solution is made froma surfactant such as Triton X-100 at a concentration of 0.1% to 10% inwater. Following defatting, the bone is immersed in acid over time toeffect demineralization. Acids which can be employed in this stepinclude inorganic acids such as hydrochloric acid and organic acids suchas peracetic acid. After acid treatment, the demineralized bone isrinsed with sterile water for injection to remove residual amounts ofacid and thereby raise the pH.

Following demineralization, the bone is subdivided into demineralizedbone particles of desired configuration and size. Useful for thesubdivision of the demineralized bone are machines or instruments knownto the arts of, e. shredding, milling, pressing, shaving, machining,extruding and/or cutting, of hard or brittle materials such as wood,plastics, soft metals, ceramics and the like. Particularly preferred aremills, including impact mills, grating mills, shearing mills and cuttingmills. Many of the preferred instruments for the subdivision of thedemineralized bone will fragment the demineralized bone, by cutting orseparating the demineralized material in direction parallel to theunderlying collagen fibers

Particularly preferred types of equipment or machine useful forshredding, cutting hard or brittle materials such as wood, plastics,soft metals that can be used to subdivide the demineralized bone includeimpact mills, grating mills, shearing mills and cutting mills. Manypreferred cutting and milling instruments and or machine will fragmentthe demineralized bone, by cutting or separating the demineralizedmaterial in direction parallel or nearly parallel to the underlyingcollagen fibers. Mills, presses and extruders are particularly useful inthis regards.

An impact mill has blunt rotors or swinging hammers that move at highspeed and subdivide the demineralized bone stock by impacting upon thebone shattering it into fragmentary particles. The bone tends to shatteralong the lines of the natural collagen bundles constituting themicrostructure of the bone. Similar mills with sharp cutting rotors tendto chop the bone into somewhat symmetric particles as opposed to thefibrous particles obtained with an impact mill. Impact speed is a factorthat influences the result. Too low a speed may cause the bone toplastically deform rather than shatter into particles as required. Thisand similar factors involved in the operation of a particular type ormodel of impact mill to provide demineralized bone fibers can beoptimized employing routine experimentation.

A shearing mill subdivides demineralized bone stock by tearing the boneapart. The tearing action tends to preferentially break the bone apartat its weakest point. The junctions between demineralized collagenbundles represent weak points and the result is the production of fibertype particles.

The spindle element of a lathe can be adapted to carry a rotary grindingwheel whose circumferential surface is studded with projecting cuttingelements. As the bone stock is pressed against the rotating wheel, thecutting elements produce fiber-type particles. In this type ofparticle-forming operation, the resulting fibrous particles are notseparated along the lines of natural collagen bundles.

Still other apparatus useful in milling bone particles according to theinvention includes mills available from IKA® Works (Wilmington, N.C.)such as the model A 10 IKA-Analytical Mill or the model M 20IKA-Universal Mill. Such mills have cooling connections and are suitablefor the grinding of hard and brittle substances with a maximum grainsize of 6-7 mm. It has been determined that a stainless steelstar-shaped cutter provides particles of a useful size. Other millingmachines useful in the practice of the invention herein include drumcutter bone mills such as those available from Tracer Designs, Inc.(Santa Paula, Calif.), e.g., its bone mill model BM1000.

A particularly effective method for subdividing demineralized bone stockis to subject the bone to pressing. The simplest pressing technique isto apply pressure to the unconstrained demineralized bone. Examplesinclude pressing the bone using a mortar and pestle, applying arolling/pressing motion such as is generated by a rolling pin, orpressing the bone pieces between flat or curved plates. These flatteningpressures cause the bone fibers to separate. Unlike the prior art methodfor making fibers from mineralized bone, pressing demineralized bone inaccordance with the present invention provides intact natural bonecollagen fibers (not composite fibers made from joined short fibersections) that can be as long as the fibers in the demineralized bonestock from which they were obtained.

Another pressing technique involves mechanically pressing demineralizedbone which is constrained within a sealed chamber having a hole (or asmall number of holes) in its floor or bottom plate. The separatedfibers extrude through the holes with the hole diameter limiting themaximum diameter of the extruded fibers. As with the unconstrainedpressing method, this constrained technique results in fibers that arelargely intact (as far as length is concerned) but separated bonecollagen bundles.

In a combined unconstrained/constrained pressing technique that resultsin longer fibers by minimizing fiber breakage, the demineralized bone isfirst pressed into an initially separated mass of fibers while in theunconstrained condition and thereafter these fibers are constrainedwithin the sealed chamber where pressing is continued.

In general, pressing of demineralized bone to provide demineralized boneparticles can be carried out at from about 1,000 to about 40,000 psi,and preferably at from about 5,000 to about 20,000 psi.

Depending on the procedure employed for producing the demineralized boneparticles, one can obtain a mass of bone particles in which at leastabout 80 weight percent, preferably at least about 90 weight percent andmost preferably at least about 95 weight percent, of the particlespossess a median length of from about 2 to about 300 mm or greater,preferably a median length of from about 5 to about 50 mm, a medianthiclmess of from about 0.5 to about 15 mm, preferably a medianthickness of from about 1 to about 5 mm, a median width of from about 2to about 35 mm, preferably a median width of from about 2 to about 20 mmand a median length to thickness ratio and/or a median length to widthratio of from about 2 to 200, preferably from about 10 to about 100. Ifdesired, the mass of bone particles can be graded or sorted intodifferent sizes, e.g., by screening, and/or any less desirable size(s)of bone particles that may be present can be reduced or eliminated.

At this time, depending upon their intended final usage, thedemineralized bone particles can be utilized as is or stored underaseptic conditions, advantageously in a lyophilized or frozen state, foruse at a later time.

The demineralized bone particles of this invention find use as, or inimplants, for a variety of orthopedic procedures where they participatein the bone healing/repair process through one or more mechanisms suchas osteogenesis, osteoinduction and osteoconduction. The demineralizedbone particles can be used as is, or formed into a variety of producttypes such as a gel, putty, or sheet. The demineralized bone particlescan optionally be admixed with one or more substances such as adhesives,fillers, plasticizers, flexibilizing agents, biostatic/biocidal agents,surface active agents, binding and bonding agents, and the like, priorto, during, or after shaping the particles into a desired configuration.Suitable adhesives, binding agents and bonding agents include acrylicresins, cellulosics, bioresorbable polymers such as polyesters,polycarbonates, polyarylates and polyfomarates. Specifically, tyrsine,polycarbonates, tyrosine polyarylates, polyglycolides, polylactides,glycolide-lactide copolymer, etc. Suitable fillers include bone powder,demineralized bone powder, hydroxyapatite, etc. Suitable plasticizersand flexibilizing agents include liquid polyhydroxy compounds such asglycerol, monacetin, diacetin, etc. Suitable biostatic/biocidal agentsinclude antibiotics, providone, sugars, etc. Suitable surface-activeagents include the biocompatible nonionic, cationic, anionic andamphoteric surfactants.

If desired, the demineralized bone particles can be modified in one ormore ways, e.g., their protein content can be augmented or modified asdescribed in U.S. Pat. Nos. 4,743,259 and 4,902,296, the contents ofwhich are incorporated by reference herein. Any of a variety ofmedically and/or surgically useful substances can be incorporated in orassociated with the bone particles either before, during or after theirformation. Thus, e.g., one or more of such substances can be introducedinto the demineralized bone particles, e.g., by soaking or immersing thebone particles in a solution or dispersion of the desired substance(s).

Medically/surgically useful substances which can be readily combinedwith the demineralized bone particles of this invention include, e.g.,collagen, insoluble collagen derivatives, etc., and soluble solidsand/or liquids dissolved therein, e.g., antiviricides, particularlythose effective against HIV and hepatitis; antimicrobials and orantibiotics such as erythromycin, bacitracin, neomycin, penicillin,polymyxin B, tetracyclines, viomycin, chloromycetin and streptomycins,cefazolin, ampicillin, azactam, tobramycin, clindamycin and gentamicin,etc.; biocidal/biostatic sugars such as dextrose, glucose, etc.; aminoacids, peptides, vitamins, inorganic elements, co-factors for proteinsynthesis; hormones; endocrine tissue or tissue fragments; synthesizers;enzymes such as collagenase, peptidases, oxidases, etc.; polymer cellscaffolds with fragments; synthesizers; enzymes such as collagenase,peptidases, oxidases, etc.; polymer cell scaffolds with parenchymalcells; angiogenic drugs and polymeric carriers containing such drugs;collagen lattices; antigenic agents; cytosketetal agents; cartilagefragments, living cells such as chondrocytes, bone marrow cells,mesenchymal stem cells, natural extracts, tissue transplants, bone,demineralized bone powder, autogenous tissues such blood, serum, softtissue, bone marrow, etc.; bioadhesives, bone morphogenic proteins(BMPs), transforming growth factor (TGF-beta), insulin-like growthfactors (IGF-1) (IGF-2); platelet derived growth factors (PDGF); growthhormones such as somatotropin; bone digestors; antitumor agents;immuno-suppressants; permeation enchancers, e.g., fatty acid esters suchas laureate, myristate and stearate monoesters of polyethylene glycol,enamine derivatives, alpha-keto aldehydes, etc.; and, nucleic acids. Theamounts of such optionally added substances can vary widely with optimumlevels being readily determined in a specific case by routineexperimentation.

The method of this invention will be better understood by way ofexample. As is the case throughout this application, all parts are byweight unless otherwise specified. The examples are provided as a meansfor explaining the invention herein and are not intended to limit theinvention in any way.

EXAMPLE 1

A right diaphysis (99 g) of human donor origin was divided lengthwiseinto four sections. The total weight of all the sections was 94 g. Thebone sections were placed in a 2-liter container along with 1410 ml of a0.6 N HCl solution. After approximately 6 hours the solution was removedand replaced with another 1410-ml portion of the acid solution. The bonesections and second aliquot of acid solution were subjected to mildvortexing with a magnetic stirrer for two days. The bone sections weredemineralized until they were completely translucent without any visiblemineralized areas indicating substantially complete demineralization.The demineralized bone sections were then rinsed with water until the pHof the rinse water was above 4.0. The demineralized bone sections werethen soaked in 70% ethanol for 1 hour. The demineralized bone sectionswere cut with scissors to fit into a model M 20 IKA-Universal Mill andprocessed in the mill for about 30 seconds to produce demineralized boneparticles in the form of fibers (yield 17.98 g, 110 cc). The fibers werethen frozen and lyophilized for about 12-15 hours.

COMPARATIVE EXAMPLE

119 g of mineralized human donor bone was milled in the milling machinedescribed in U.S. Pat. No. 5,607,269 to provide a quantity ofmineralized bone particles in the form of fibers. The mineralized fiberswere then subjected to a demineralization process described as follows.Allogenic cortical bone is placed in a reactor. A 0.6 N solution of HC1at 15 ml per gram of bone is introduced into the reactor, thedemineralization reaction proceeding for 1 to 2 hours. Followingdrainage of the HCl, the bone is covered with 0.6 N HCl/20 ppm-2000 ppmnonionic surfactant solution for 24 to 48 hours. Following drainage ofthe HCL/surfactant solution, 0.6 N HCl at 15 ml per gram of bone isintroduced into the reactor, the demineralization reaction proceedingfor another 40 to 50 minutes resulting in substantially completedemineralization of the starting cortical bone. Following drainagethrough a sieve, the demineralized bone is rinsed three times with waterfor injection at 15 ml per gram bone weight with the water for injectionbeing replaced at 15-minute intervals. Following drainage of the waterfor injection, the demineralized bone is covered with alcohol andallowed to soak for at least 30 minutes. The alcohol is then drained andthe bone is rinsed with water for injection. The demineralized bone isthen subdivided in the bone milling apparatus of U.S. Pat. No. 5,607,269to yield a mass of demineralized bone particles of fibrousconfiguration. The demineralized bone elements are then drained andtransferred to a lyophilization tray and frozen at −70° C. for at least6 hours. The demineralized bone particles are then lyophilized followingstandard procedures for 24 to 48 hours. After drying, the demineralizedbone particles are sorted for size. The yield of substantially fullydemineralized bone particles made following this procedure, as measuredbefore drying, was yield 15.27 g, 75 cc.

The following table compares the yields between the method of Example 2illustrating the present invention and the prior art method illustratedin the Comparative Example:

Amount of Wt. % Yield of Product Demineralized Starting DemineralizedBone Particles Whole bone Bone Particles Based on Wt. of Wt. (g) wt. (g)vol. (cc) Whole Bone Example 2 99 17.98 110 18.2 Comparative 119 15.2775 12.8 Example

As these data show, the method of this invention in whichdemineralization of the whole bone precedes its subdivision intodemineralized bone particles (Example 1) yielded almost 50 wt. % moreuseful product than that resulting from the prior art method in whichdemineralization is conducted only after the whole bone has beensubdivided into mineralized bone particles (Comparative Example).

EXAMPLE 2

Substantially fully demineralized fibula cross sections of about 25 mmin length were initially pressed between two flat plates of a Carverpress[?] at pressures ranging from 5,000 up to about 20,000 psi. Thisfirst pressing operation flattened the bone sections and began toseparate their collagen bundles into fibers. This material was then[“fluffed up”] and pressed again employing similar pressures as before.The pressing operation was again repeated yielding a mass of coarsebundles of fibers that were not completely separated from each other.The yield of fibers was about 50 wt. % based on the volume of the[starting demineralized bone sections] and many of the fibers possessedlengths that were nearly as great as the natural fibers of the bonestock. The fibers ranged in length from 10-15 mm, with some fibers inthe range of from 20-25 mm, and possessed diameters of about 2 mm.Material that was not in fiber form remained in bundled fiber clumps.

The fibers were further subdivided in an impact mill for [30 seconds]resulting in a reduction of the diameters of many of the fibers andfiber bundles without, however, significantly reducing their length.Thus, the fibers continued to fall within the aforesaid range of lengthbut their diameters were now within the range of from about 0.5 to about2 mm.

EXAMPLE 3

Demineralized fibula cross sections, about 25 mm in length, were placedin a series of 29 mm diameter press cells possessing single orifices intheir bottoms having diameters of 1, 2 and 3 mm, respectively. Underpressures of from 5,000-10,000 psi, the demineralized bone sectionssubdivided into fibers which extruded through the orifices. Yields ofdemineralized fiber were on the order of nearly 100 wt. % in almostevery case; little or no bone remained in the cells.

EXAMPLE 4

A cell pressing procedure similar to that of Example 4 was carried outon demineralized bone sections of from 4 to 8 mm in length in a 29 mmdiameter press cell having a single orifice of 0.75 mm diameter. At apress load of 5,000 to 10,000 psi, the bone sections subdivided intofibers that ranged in length from 25 to 50% of the length of the bonesections from which they were obtained. Yield of fiber was about 50wt.%. The fibers ranged in length from about 1-5 mm and possessed adiameter of about 0.5 mm.

EXAMPLE 5

Substantially fully demineralized fibula cross sections of about 25 mmin length were pressed in the press cells described in Example 4. Atpressures ranging from 5,000 to 10,000 psi, the bone sections subdividedinto fibers having the dimensions set forth in the following table:

Approximate Diameter of Press Approximate Length Diameter of Fibers,Cell Orifice, mm of Fibers, mm mm 1 1-5 1.5-2  2 1-5 1.75-275 3 1-5 3-3.5

EXAMPLE 6

The pressing operations described in Example 5 were substantiallyrepeated but were preceded by a preliminary pressing carried out in aCarver press at 15,000 psi. The resulting demineralized bone fiberspossessed smaller diameters, and consequently, greater length todiameter ratios, than the fibers obtained in Example 6.

EXAMPLE 7

Substantially fully demineralized whole fibula shafts were subdividedinto fibrous particles employing a Tracer (rotary grater) mill. Fiberlength was about 5 mm, diameter was about 0.5 mm and fiber yield wasabout 70 wt. %.

EXAMPLE 8

Example 8 was repeated but with fibula sections of 4-8 mm in length.Fiber length was about 3-5 mm, diameter was about 0.5 mm and fiber yieldwas about 50 wt %.

EXAMPLE 9

A model M5A Fitzpatrick Mill was employed to subdivide substantiallyfully demineralized bovine bone chips of 4-10 mm into fibrous particleshaving a length of about 1-2 mm and a diameter of about 0.2-0.7 mm in ayield of about 70 wt. %.

EXAMPLE 10

Example 9 was repeated but employing a model M 20 IKA-Universal Mill tosubdivide the demineralized bovine bone chips. The fibers in thefiber-fraction produced by the mill had a length of about 1-2 mm, adiameter of about 0.5-1 mm and the fiber yield was about 10%.

EXAMPLE 11

Example 9 was repeated but employing a Megatron homogenizer (Glen MillsInc., Maywood, N.J.). The resulting fibers, produced in a yield of about70 wt. %, possessed a length of about 1-3 mm and a diameter of about0.2-0.5 mm.

1-25. (canceled)
 26. A method of making demineralized bone particles,comprising: demineralizing whole bone, the whole bone comprisingcollagen fibers; and thereafter subdividing the demineralized bone bypressing the demineralized bone into demineralized bone particlescomprising intact collagen fibers.
 27. A method as recited in claim 26,wherein the intact collagen fibers are not joined from fiber sections.28. A method as recited in claim 26, wherein at least one of the intactcollagen fibers has a length equal to at least one of the collagenfibers of the whole bone.
 29. A method as recited in claim 26, whereinthe intact collagen fibers range in length from 10-15 mm.
 30. A methodas recited in claim 26, wherein the intact collagen fibers range inlength from 20-25 mm.
 31. A method as recited in claim 26, whereinpressing the demineralized bone into demineralized bone particlescomprises: performing a first pressing by applying pressure ranging fromabout 5,000 to about 20,000 psi; and performing a second pressing byapplying pressure ranging from 5,000 to about 10,000 psi.
 32. A methodas recited in claim 26, wherein pressing the demineralized bonecomprises pressing the demineralized bone between two plates of a Carverpress.
 33. A method as recited in claim 26, wherein pressing thedemineralized bone comprises applying a pressure from about 1,000 toabout 40,000 psi.
 34. A method as recited in claim 26, wherein pressingthe demineralized bone comprises applying a pressure from about 5,000 toabout 20,000 psi.
 35. A method as recited in claim 26, whereindemineralizing the whole bone comprises defatting the whole bone in anaqueous solution of ethanol, the solution comprising about 60% to about90% alcohol by weight.
 36. A method as recited in claim 26, whereindemineralizing the whole bone comprises defatting the whole bone in asolution of comprising Triton X-100 at a concentration of 0.1% to 10% inwater.
 37. A method as recited in claim 26, wherein demineralizing thewhole bone comprises immersing the whole bone in peracetic acid.
 38. Amethod as recited in claim 26, wherein a yield of demineralized boneparticles is greater than that obtained by subdividing the whole boneinto mineralized bone particles and thereafter demineralizing themineralized bone particles to provide demineralized bone particles. 39.A method as recited in claim 26, wherein at least about 80 weightpercent of the particles have a median length to width ratio of fromabout 2 to
 200. 40. A method of making demineralized bone particles,comprising: demineralizing whole bone, the whole bone comprisingcollagen fibers; and thereafter subdividing the demineralized bone bypressing the demineralized bone into demineralized bone particlescomprising intact collagen fibers, wherein at least one of the intactcollagen fibers have a length equivalent to that of at least one of thecollagen fibers of the whole bone and the demineralized bone particlesare separated into bone collagen bundles.
 41. A method of makingdemineralized bone particles, comprising: demineralizing whole bone, thewhole bone comprising collagen fibers; thereafter subdividing thedemineralized bone by pressing the demineralized bone into demineralizedbone particles comprising intact collagen fibers; and forming thedemineralized bone particles into one of a group consisting of a gel, aputty or a sheet.
 42. A method as recited in claim 41, furthercomprising admixing the demineralized bone particles with at least oneof a group consisting of adhesives, fillers, plasticizers, flexibilizingagents, biostatic/biocidal agents, surface active agents, and bindingand bonding agents.
 43. A method as recited in claim 41, furthercomprising admixing the demineralized bone particles with at least oneof a group consisting of acrylic resins, cellulosics, bioresorbablepolymers, polyesters, polycarbonates, polyarylates, polyfomarates,tyrsine, polycarbonates, tyrosine polyarylates, polyglycolides,polylactides, glycolide-lactide copolymer, bone powder, demineralizedbone powder, hydroxyapatite, liquid polyhydroxy compounds, glycerol,monacetin, diacetin, antibiotics, providone, sugars, and amphotericsurfactants
 44. A method as recited in claim 41, wherein thedemineralized bone particles are formed into a sheet.